Macrophages Modulate Optic Nerve Crush Injury Scar Formation and Retinal Ganglion Cell Function

Yuan Liu; Xiangxiang Liu; Christopher A. Dorizas; Zixuan Hao; Richard K. Lee

doi:10.1167/iovs.65.10.22

Abstract

Purpose: Optic nerve (ON) injuries can result in vision loss via structural damage and cellular injury responses. Understanding the immune response, particularly the role of macrophages, in the cellular response to ON injury is crucial for developing therapeutic approaches which affect ON injury repair. The present study investigates the role of macrophages in ON injury response, fibrotic scar formation, and retinal ganglion cell (RGC) function.

Methods: The study utilizes macrophage Fas-induced apoptosis (MaFIA) mice to selectively deplete hematogenous macrophages and explores the impact macrophages have on ON injury responses. Histological and immunofluorescence analyses were used to evaluate macrophage expression levels and fibrotic scar formation. Pattern electroretinogram (PERG) recordings were used to assess RGC function as result of ON injury.

Results: Successful macrophage depletion was induced in MaFIA mice, which led to reduced fibrotic scar formation in the ON post-injury. Despite an increase in activated macrophages in the retina, RGC function was preserved, as demonstrated by normal PERG waveforms for up to 2 months post-injury. The study suggests a neuroprotective role for macrophage depletion in ON damage repair and highlights the complex immune response to ON injury.

Conclusions: To our knowledge, this study is the first to use MaFIA mice to demonstrate that targeted depletion of hematogenous macrophages leads to a significant reduction in scar size and the preservation of RGC functionality after ON injury. These findings highlight the key role of hematogenous macrophages in the response to ON injury and opens new avenues for therapeutic interventions in ON injuries. Future research should focus on investigating the distinct roles of macrophage subtypes in ON injury and potential macrophage-associated molecular targets to improve ON regeneration and repair.

Optic nerve (ON) injuries, caused by traumatic, glaucomatous, or other neurodegenerative conditions, are of significant clinical importance and is an area of prominent research for central nervous system (CNS) regeneration and repair.¹ The ON, which consists of the axons of retinal ganglion cells (RGCs), extends from the CNS and is vital for vision. ON injury can lead to partial or complete loss of vision, which has a profound impact on the quality of life of those affected.² Moreover, comprehension of the ON’s response to injury produces valuable insights into CNS repair mechanisms and the development of therapeutics to ameliorate vision loss.³^,⁴ As part of the CNS, the ON exhibits complex responses to brain and spinal cord injury, including inflammation, scar formation, and cellular responses.⁵^–⁷ Research into ON injuries not only addresses a critical clinical need, but also broadens our understanding of CNS regeneration and repair processes, which is important for advancing the field of neurobiology.⁸^,⁹

Within the field of CNS injuries, macrophages are recognized as key players influencing many pathophysiological processes.¹⁰^–¹² These immune cells emerge from both resident microglia and infiltrating peripheral sources and play important roles in phagocytosis, secretion of inflammatory mediators, and modulation of the injury microenvironment.¹³^,¹⁴ Understanding the function of macrophages, which exhibit both pro- and anti-inflammatory states, and their specific contributions to neuroinflammation, neuronal survival, and tissue repair is crucial for developing therapeutic approaches in ON injuries.¹⁵^,¹⁶ However, in the ON injury process, questions are unanswered regarding how macrophages impact the survival and function of RGCs and their involvement in post-injury ON recovery and scarring.¹¹ Significant interest exists in how macrophage activity can be modulated for neuronal repair. The effectiveness of such approaches in the ON injury mouse model requires further investigation.¹⁷

In this study, we used macrophage Fas-induced apoptosis (MaFIA) mice to selectively deplete hematogenous macrophages to investigate the role of macrophages in the ON injury response, fibrotic scar formation, and RGC function. By combining histological analysis with functional assessments, we provide new insights into this complex interplay between immune responses and ON injury. This research has the potential to identify novel therapeutic approaches to ON injury and contribute to the broader goal of improving neural regeneration and recovery in CNS disorders.

Methods

Mice

All animal procedures were in accordance with University of Miami IACUC and National Institutes of Health (NIH) guidelines. Furthermore, all studies conformed to the standards in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The MaFIA mice were purchased from the Jackson Laboratory (JAX 005070; Bar Harbor, ME, USA). The MaFIA mice enable the inducible and reversible triggering of apoptosis in macrophages and dendritic cells by utilizing the mouse colony stimulating factor 1 receptor promoter (Csf1r) to activate the expression of a mutant form of human FK506 binding protein 1A, 12kDa. Additionally, the transgene facilitates the fluorescent tagging of cells expressing Csf1r. The MaFIA mice and wild-type (WT) mice used in our experiments were 8 to 12 weeks old and all mice were in a C57BL/6 genetic background. The mice were bred and maintained in the University of Miami animal facility and housed under standard conditions of temperature and humidity with a 12-hour light/dark cycle and free access to food and water. For all surgical procedures, the mice were anaesthetized with 100 mg/kg ketamine and 15 mg/kg xylazine intraperitoneally, and the eyes were topically anesthetized using 0.5% proparacaine hydrochloride. Eye ointment containing erythromycin was applied postoperatively to protect the cornea and prevent conjunctival wound infection.

Optic Nerve Crush

Optic nerve crush (ONC) injury was performed as described previously.⁵ The 8 to 12-week-old mice were anesthetized and monitored until loss of response to toe-pinch. Under a binocular operating microscope, a superior conjunctival peritomy of the left eye was performed on topically and systemically anesthetized mice. The superior ocular muscles were reflected aside, and the ON was exposed at its exit from the eye globe. Dumont #5 forceps (FST) were used to crush the ON approximately 1 to 2 mm behind the optic disk without damaging the retinal vessels or the blood supply.

Macrophage Depletion

Lyophilized AP20187 was dissolved in 100% ethanol at a concentration of 13.75 mg/mL or as a 1-mM stock solution in ethanol and stored at −20°C. For in vivo use, peritoneal injections were prepared from the 13.75 mg/mL ethanol stock diluted to 0.55 mg/mL for an injection solution consisting of 4% ethanol, 10% PEG-400, and 1.7% Tween in water. All injections were administered to mice within 30 minutes of dilution into the injection solution. The volume of injection solution was adjusted according to mouse body weight to deliver 10 mg AP20187 Fas dimerizer per kg per mouse. AP20187 injection was performed for 5 consecutive days after the ONC (Fig. 1).

Figure 1.

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Timeline of the macrophage depletion procedure.

Histology and Immunofluorescence

Mice were anesthetized and perfused transcardially with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS for 5 minutes to fix the tissue. The ONs were dissected and post-fixed with 4% PFA in PBS overnight and incubated in 30% sucrose overnight (4°C). For histological sectioning, ON samples were embedded in OCT compound (Tissue-Tek; Sakura Finetek, USA) and serial longitudinal sections were cut using a cryostat. ONs were cut at 8 µm and mounted onto Platinum Line Microscope Slides (Mercedes Medical, Germany), and stored at –20°C.

To prepare liver and spleen sections for immunofluorescence, animal tissues were immersed in 4% PFA overnight. Following fixation, the tissues were embedded in paraffin, sliced into 10-µm sections, and affixed onto glass slides. Before staining, the sections underwent deparaffinization in xylene and successive alcohol washes. Subsequently, the slides were subjected to antigen retrieval using Rodent Decloaker (Biocare Medical, Pacheco, CA, USA) reagent at 95°C.

For staining, nonspecific antibody binding was blocked for 20 minutes at room temperature with Rodent Block M (Biocare Medical). Histologic sections were then incubated with primary antibodies (see the Table), diluted in PBS with 0.5% Triton X-100, at 4°C overnight in a humidified box. After washing in PBS 3 times for 5 minutes each, the sections were incubated with species-specific fluorescent secondary antibodies for 1 hour at room temperature (see the Table). Finally, the sections were cover-slipped with Vecta shield (Vector) fluorescent mounting medium containing DAPI (Vector Laboratories H-1200). Imaging was performed with a Leica TSL AOBS SP5 confocal microscope (Leica Microsystems).

Table.

View Table

Antibodies Used in This Study

Quantification of Ganglion Cell Layer Neurons in the Retina

Whole mount retinas were harvested on day 10 after ONC. Eyeballs were fixed with 4% PFA in PBS (1 × PBS) solution for 1 hour at room temperature. For flat mounts, the cornea and crystalline lens were removed, and eyecups were fixed in 4% PFA for an additional 2 hours. The entire retina was then carefully dissected from the eyecup and immersed in 30% sucrose overnight (4°C). Retinas were permeabilized with 0.5% Triton X-100 in 1 × PBS for 1 hour and then blocked with Rodent Block M for 1 hour. Whole mount retinas were incubated overnight at 4°C in chicken GFP antibody (AB13970; see the Table) diluted in 1 × PBS with 0.1% Triton X-100 (pH = 7.4). After 3 washes with PBS, the retinas were incubated with donkey anti-chicken secondary antibody (Jackson ImmunoResearch; see the Table) for 2 hours at room temperature in PBS. Flat-mounted retinas were washed in PBS 3 times for 15 minutes each, and then mounted on glass slides (with the RGC layer facing up).

Four radial cuts were made from the edge to the equator of the retina to flatten the retina. Retinas were mounted with Vecta shield fluorescent mounting medium containing DAPI and cover slipped. In each quadrant, six discontinuous images were taken from the central region of retina to the periphery using a Leica TSL AOBS SP5 confocal microscope (20 × objective; Leica Microsystems, Exton, PA, USA). The number of GFP-positive macrophages in each image was counted with ImageJ software.

Pattern Electroretinogram

Pattern electroretinogram (PERG) recording was performed as described by Porciatti et al.¹⁸ During PERG recording, the mice anesthetized with intra-peritoneal ketamine/xylazine were placed on a feedback-controlled heating pad (TCAT-2LV; Physitemp Instruments, Inc., Clifton, NJ, USA) to maintain a constant body temperature at 37°C. For PERG recording, a semicircular silver loop electrode was placed on the topically anesthetized cornea. Reference and ground electrodes were placed subcutaneously on the back of the head and the base of the tail, respectively. Anesthetized mice received a stimulus of contrast bars (field area, 69.4 degrees × 63.4 degrees; mean luminance, 50 cd/m²; spatial frequency, 0.05 cycles/deg; contrast, 98%; temporal frequency, 1 hertz [HZ]) at a distance of 20 cm. Three independent test trials composed of 300 measurements was recorded for each eye and the data was processed using Sigmaplot (version 11.2; Systat Software, Inc., San Jose, CA, USA). The peak to trough amplitude in a time window of 50 to 300 ms was automatically measured and analyzed by the PERG software.

The PERG responses were superimposed automatically to compare for consistency and then averaged. The major positive (P1) and negative waves (N2), the sum of their absolute values (peak-to-trough amplitude), and the peak latency of the major positive wave (P1) were automatically analyzed, calculated, and graphed using MATLAB software (MathWorks).

Quantification

Quantification of immunohistochemical images were performed by unbiased masked observers using ImageJ software.

To quantify GFP, CD3, and CD11b positive cells in the liver and spleen, three high magnification images (40 × objective) were taken for each animal. The cells were counted only if they exhibited positive staining for the respective markers and were accompanied with DAPI+ staining. Each experimental group consisted of three independent animals.

To quantify the number of CD68⁺ cell density at the ON injury site, 2800 pixel × 1200 pixel grids were generated from the entire image (40 × objective) which included the entire injury site. Only CD68⁺ cells accompanied with DAPI⁺ were counted and cells touching the left and bottom limits of a square were disregarded. The number of CD68⁺ cells in each section was normalized to the area of ON and for each animal the counts from each section were averaged.

For each animal, the fibrotic scar area was measured according to GFAP-negative staining regions by image J software and averaged from each section.

Statistical Analysis

All data are represented as mean ± standard error. The statistical significance comparing GFP, CD3, and CD11b cells in the liver and spleen, macrophage numbers in ON, and scar size was assessed using an unpaired t-test. The GFP⁺ monocyte numbers in different layers of the retina were compared using 1-way ANOVA with repeated measures using the Tukey's post hoc test. PERG results were compared using 2-way ANOVA with the Tukey's post hoc test. The t-test and ANOVA were performed using GraphPad prism software version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Macrophage Depletion in MaFIA Mice

Mice were euthanized 14 days ON post-crush, during which a significant macrophage accumulation in the injury area was observed.⁵ Prior studies demonstrated the re-establishment of depleted GFP⁺ cells in tissues after completing the dimerizer treatment protocol.¹⁹ Given the 9-day gap between the last AP20187 injection and mouse euthanization, it was crucial to verify the distribution and quantity of macrophage depletion in MaFIA mice using immunofluorescence with anti-GFP antibody.

A notable presence of GFP-positive macrophages was evident in the spleens of MaFIA mice without AP20187 treatment (Figs. 2A, 2B). The highest concentration of GFP⁺ cells in mock-treated mice was observed in the red pulp areas and marginal zones of the follicles. AP20187 treatment to induce Fas-based macrophage depletion significantly reduced the number of GFP⁺ cells across all spleen regions, with most residual GFP⁺ cells concentrated in the red pulp (Figs. 2C, 2D). CD3-positive T cells observed in C57BL/6 (BL/6) WT mice (Supplementary Figs. S1E, S1F) and non-macrophage-depleted MaFIA mice (Supplementary Figs. S2E, S2F) were reduced in AP20187-treated mice (Figs. 2H, 2I). CD11b-positive myeloid-lineage cells were noted in WT mice (Supplementary Figs. S1I S1J) and MaFIA mice without AP20187 treatment (see Figs. 2I, 2J), with a significant reduction observed 9 days after the last AP20187 injection (Figs. 2K, 2L).

Figure 2.

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Immunostaining and hematoxylin and eosin (H&E) staining results of MaFIA mice spleen. GFP staining of MaFIA mice without AP20178 injection (A), higher magnification image of A (B), and MaFIA mice with AP20187 staining (C), and higher magnification image of C (D). The number of GFP-positive monocytes in MaFIA mice decreased after AP20187 injection (R red pulp and W white pulp). White arrows point the GFP+ monocytes. CD3-positive T cells observed in non-macrophage-depleted MaFIA mice (E) and higher magnificent image (F). MaFIA mice with AP20187 injection showed significant loss of CD3 positive cell (G) and higher magnificent image (H). CD11b-positive myeloid-lineage cells were noted in MaFIA mice without AP20187 treatment (I), higher magnification image of I (J), with a significant reduction observed in macrophage AP20187 treated MaFIA mice (K), higher magnification image of K (L). H&E staining of MaFIA mice spleen. MaFIA mice without AP20187 injection had normal structure (M, N). MaFIA mice with AP20187 injection showed severe disruption of follicular morphology (O, P). Scale bar = 10 × magnification, 100 µm; 40 × magnification, 20 µm. (Q) Quantification results of GFP positive cells in the spleen of MaFIA mouse without and with AP20187 injection (n = 3, Student’s t-test, ***P < 0.005). (R) Quantification results of CD3 positive cells in the spleen of MaFIA mouse without and with AP20187 injection (n = 3, Student’s t-test, ***P < 0.005). (S) Quantification results of CD11b positive cells in the spleen of MaFIA mouse without and with AP20187 injection (n = 3, Student’s t-test, ***P < 0.005).

Histological evaluation revealed extensive extramedullary hematopoiesis in the spleens of macrophage/DC-depleted mice. Spleens exhibited islands of extramedullary hematopoiesis with megakaryocytosis and severe disruption of follicular morphology (Figs. 2M–2P).

Nine days after the last AP20187 treatment, no GFP⁺ and CD11b⁺ cells were observed in MaFIA mice, indicating the macrophage count did not recover in the liver (Figs. 3C, 3D, 3K, 3L). Islands of extramedullary hematopoiesis were also observed in the livers of depleted MaFIA mice, although the extent of tissue morphology disruption was less severe than in the spleen (Figs. 3M–3P).

Figure 3.

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Immunostaining and hematoxylin and eosin (H&E) staining results of MaFIA mice liver. GFP staining of MaFIA mice without AP20187 injection (A), higher magnification image of A (B), and MaFIA mice with AP20187 injection staining (C), and higher magnification image of C (D). White arrows point to the GFP+ monocytes. CD3-positive T cells were observed in non-macrophage-depleted MaFIA mice (E, F) and not in macrophage-depleted mice (G, H). CD11b-positive myeloid-lineage cells were noted in MaFIA mice without AP20187 treatment (I, J), with a significant reduction observed in macrophage AP20187 treated MaFIA mice (K, L). H&E staining of MaFIA liver. MaFIA mice without AP20187 injection had normal structure (M, N). MaFIA mice with AP20187 injection showed structure disruption (O, P). Scale bar = 10 × magnification, 100 µm; 40 × magnification, 20 µm. (Q) Quantification results of GFP positive cells in the liver of MaFIA mouse without and with AP20187 injection (n = 3, Student’s t-test, ***P < 0.005). (R) Quantification results of CD3 positive cells in the liver of MaFIA mouse without and with AP20187 injection (n = 3, Student’s t-test, ***P < 0.005). (S) Quantification results of CD11b positive cells in the liver of MaFIA mouse without and with AP20187 injection (n = 3, Student’s t-test, *P < 0.05).

Macrophages are Depleted in the Optic Nerve

To investigate the involvement of hematogenous macrophages in the formation of fibrotic scars, we depleted these macrophages by administering AP20187 injections to MaFIA mice following ONC. We assessed the macrophage count in the ON post-crush. Following AP20187 treatment, MaFIA mice exhibited a significantly lower CD68 positive macrophage presence in the middle and distal areas of the ON compared to BL/6 mice (Figs. 4A1–4D3, 4G, 4H). GFAP-positive astrocytes activation were also reduced in the MaFIA with AP20187 injection (Figs. 4A1–4D3). However, the disparity in macrophage numbers at the injury site between MaFIA and control BL/6 mice did not show statistical significance (Figs. 4E1–4F3, 4I).

Figure 4.

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Immunostaining images and quantitation results of macrophages expression in the optic nerve. Staining outcomes depicting the optic nerve of BL/6 mice following ONC injury reveal the activation of CD68 macrophages and GFAP astrocytes in distal, middle and injured area, respectively, (A1-A3, C1-C3, E1-E3). Staining outcomes of MaFIA mice subjected to optic nerve crush injury post AP20187 injection in the distal, middle, and injured areas, respectively (B1-B3, D1-D3, F1-F3). Quantitative analysis demonstrates a noteworthy reduction in the number of CD68-positive cells in the distal and middle areas of optic nerve of MaFIA mice following crush injury (G, H). No significant difference is observed in the number of CD68-positive cells in the injured area of the optic nerve between BL/6 mice and MaFIA mice with AP20187 injection after crush injury (I) (n = 4, Student’s t-test, *P < 0.05, Scale bar = 50 µm). GFAP staining of optic nerve after injury of MaFIA mice with AP20187 injection (J) and BL/6 mice (K), GFAP-negative staining area was marked with a white dotted line. Quantification results demonstrated significant reduction of GFAP-negative scar size in MaFIA mice with AP20187 injection (L) (n = 5, Student's t-test, *P < 0.05, Scale bar = 100 µm).

Macrophage Depletion Reduces Fibrotic Scar Formation

Based on the earlier findings,⁵ we selected the 14th day after ONC as the observation time to examine the relationship between the reduction of hematogenous macrophages and alterations in fibrotic scarring. Our investigation revealed that macrophage depletion resulted in a reduction of ON scar size. The average fibrotic area in each ON section was significantly diminished following AP20187 treatment compared to the BL/6 group (Figs. 4J–4L).

Macrophage Activation in the Retina and Preservation of RGC Function

Axonal injury within the ON initiates the demise and degeneration of mature RGCs. The RGC somata become disconnected from their source of neurotrophic factors, which are retrogradely transported from the synaptic terminals in the brain. To further explore the impact of this, we investigated the distribution of GFP⁺ macrophages in the retina and assessed whether their depletion could safeguard RGC function post-injury. Our findings reveal a notable increase in the number of activated macrophages in the injured eye of MaFIA mice treated with AP20187 compared to the non-injured eye of MaFIA mice with the same treatment, as well as the injured and non-injured eyes of MaFIA mice without AP20187 (Fig. 5).

Figure 5.

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Immunostaining images and quantification results of monocytes in the retina. Left retina of MaFIA mice without AP20187 injection in the peripheral, middle, and central areas, respectively (A1-F1 column), right retina of MaFIA mice without AP20187 injection in the peripheral, middle, and central areas, respectively (A2-F2 column), retina of the crushed left eye of MaFIA mice with AP20187 injection in the peripheral, middle, and central areas, respectively (A3-F3 column) and the right eye of MaFIA mice with AP20187 injection in the peripheral, middle, and central areas of the retina, respectively (A4-F4 column) demonstrated activation of GFP positive monocytes in both inner and outer plexiform layer. Quantification results the demonstrated crushed eye of MaFIA mice with AP20187 injection had significantly larger number of activation monocytes in inner and outer plexiform layer (G-L) (n = 4, 1-way ANOVA, *P < 0.05, Scale bar = 57.9 µm).

Furthermore, we utilized pattern electroretinography to evaluate the function of RGCs. Remarkably, MaFIA mice injected with AP20187 exhibited a normal PERG waveform even 2 months after the injury (Fig. 6B). WT BL/6 mice lost PERG signal 2 months after injury (Fig. 6D). The PERG amplitude ratio between the injured and non-injured eyes in macrophage-depleted MaFIA mice was significantly higher than in control BL/6 mice, demonstrating preserved RGC function in macrophage depleted mice (Fig. 6E).

Figure 6.

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PERG results of BL/6 mice and MaFIA mice after injury. Baseline assessment of the eye in MaFIA mice (A). MaFIA mice eye subjected to crush injury with AP20187 injection 2 months post-crush (B). Baseline evaluation of the eye in BL/6 mice (C). BL/6 mice eye following crush injury 2 months post-crush (D). Ratio of PERG amplitude between the crushed and non-crushed eyes (E). MaFIA mice exhibit a significantly higher ratio at both one and two months after the crush injury (2-way ANOVA, *P < 0.05).

Discussion

In this study, we investigated the mechanism of fibrotic scar development following ON injury by focusing on the role of macrophages in ON regeneration. By using MaFIA mice, we have demonstrated that macrophage depletion significantly reduces the population size of macrophages in the ON, particularly in its middle and distal regions. This decrease substantially reduces fibrotic scarring, emphasizing the important role of hematogenous macrophages in the injury response and scarring following ON injury. Additionally, this study reveals the neuroprotective implications of macrophage depletion. The depletion of macrophages maintained RGC function in ON injury, as demonstrated by the maintenance of normal PERG waveforms up to 2 months ON post-injury compared to the loss of PERG activity after ON post-injury in WT mice.

We previously demonstrated that macrophages after ON injury originate from two distinct sources within the monocyte/phagocyte system.²⁰ Microglia, which are the CNS’s primary resident immune cells, respond to ON damage and reach peak activity around 2 to 3 days after injury.⁵^,²¹ Blood-derived monocytes, acting as macrophages, begin to accumulate at the injury site around 3 days after injury, peaking approximately a week later.⁵

The present study used MaFIA mice to visualize macrophages from the peripheral hematogenous circulation. After AP20187 injection and induction of macrophage depletion via a Fas-activated apoptotic pathway, a significant reduction in GFP⁺ labeled cells was observed across all splenic regions, with most residual cells concentrated in the red pulp. The significant reduction in CD11b-positive myeloid lineage cells and absence of CD3-positive T-cells in AP20187-treated mice demonstrates macrophage depletion. The absence of GFP⁺ and CD11b⁺ cells in the livers of MaFIA mice 9 days post-treatment suggest no regeneration of macrophage populations. The use of this transgenic mouse effectively eliminated blood-derived macrophages, thereby providing a good model and basis for further investigation of their role in ON injury.

Previous research has demonstrated that the ON injury process is associated with the activation of monocytes and macrophages.¹¹^,²⁰ During the first 3 days of fibrosis formation, inflammation is involved in clearing debris, with blood-derived monocytes or macrophages recruited to the injury site. These monocytes then release pro-inflammatory cytokines, leading to the formation of scar tissue characterized by reactive glial cells, fibroblast migration, and extracellular matrix formation.²²^–²⁴ However, resident and circulating macrophages have different roles in tissue repair.²⁵^,²⁶ Research into acute lung injury has shown that recruited macrophages – as distinguished from resident ones – produce inflammatory cytokines and increase glycolysis.²⁷^–²⁹ As a result, it is suggested that inhibiting the recruitment of hematogenous macrophages could reduce fibroblast accumulation in the ONC site.

The findings in the present paper align with our previous studies.¹¹ The results suggests that following macrophage depletion, the size of the scar was significantly reduced in comparison to controls. This demonstrates a decrease in both cellular and extracellular matrix accumulation at the site of ONC following hematogenous macrophage depletion. Together, these results and the reduced hematogenous macrophage expression in the ON observed in the present study suggest that the depletion of these macrophages leads to a smaller ONC scar size, which may promote the regeneration of axons and improve functional recovery in an environment that supports cellular growth.¹¹

Previous studies demonstrate that M1 (pro-inflammatory) macrophages play a significant role in astrogliosis after spinal cord injury.³⁰ They are involved in a rapid and sustained M1 response at the site of injury.³¹^,³² It is possible that eliminating M1 macrophages may delay or diminish the astrocyte response during the ONC’s acute phase. M2 macrophages are induced within 3 to 7 days after spinal cord injury and can reduce spinal cord inflammation and phagocytose tissue debris, which is critical for nerve regeneration and matrix remodeling.³³^,³⁴ The balance of M1 and M2 macrophages at the injury site is involved in the development and progression of the secondary inflammatory response.³⁵^–³⁷

This study highlights the dynamic nature of macrophage phenotypes and their critical role in the inflammatory response and repair processes in CNS injury. Future research is needed to investigate the effects of different subtypes of macrophages on gliosis in both the acute and chronic phases following ONC.

Interestingly, the maintenance of RGC function (measured by PERG activity) following macrophage depletion, as observed in our study, provides further evidence for the neuroprotective potential of modulating macrophage activity. These findings are significant given the complex interplay between macrophages and RGCs in the retina.³⁸

Macrophages are present in the inner and outer layers of the retina, where they have a key role in maintaining retinal homeostasis and responding to injury or disease.³⁹^–⁴¹ Their functions include phagocytosis of debris, secretion of neurotrophic factors, and modulation of inflammatory responses, all of which are critical for the health and function of RGCs.⁴²^–⁴⁴ Recent research has shown that macrophages secrete cytokines that significantly enhance neuronal survival and regeneration after CNS injury.⁴⁵^,⁴⁶ The potential of macrophages to switch between pro-inflammatory and anti-inflammatory states suggests that they can either exacerbate or alleviate neuronal damage.⁴⁷^–⁴⁹ Further studies which focus on the distribution and identity of subtypes of macrophages in the retina and their correlation with RGC function are needed.

Conclusions

To our knowledge, this study is the first to use MaFIA mice to demonstrate that targeted depletion of hematogenous macrophages leads to a significant reduction in ONC scar size and the preservation of RGC function after ON injury. These findings highlight the key significance of hematogenous macrophages in the response to ON injury and opens new avenues for therapeutic interventions in ON injuries. Future research will focus on investigating the distinct roles of macrophage subtypes in ON injury and identifying molecular targets to improve ON regeneration.

Acknowledgments

The Bascom Palmer Eye Institute is supported by NIH Center Core Grant P30EY014801 and a Research to Prevent Blindness Unrestricted Grant (GR004596-1). R.K.L. is supported by the Walter G. Ross Foundation. X.X.L. is supported by the National Natural Science Foundation of China (No: 82201170). This work was partly supported by the Guitierrez Family Research Fund and the Camiener Foundation Glaucoma Research Fund.

This study was presented as a poster at the Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, New Orleans, Louisiana, United States, May 2023.

Disclosure: Y. Liu, None; X. Liu, None; C.A. Dorizas, None; Z. Hao, None; R.K. Lee, None

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Figure 1.

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Timeline of the macrophage depletion procedure.

Figure 2.

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